NF1 mutations as biomarker of response to immune checkpoint blockades for lung adenocarcinoma patients

Little is known about immune checkpoint inhibitors (ICI) response of NF1-mutated lung adenocarcinomas. 341/4,181 (8.2%) TCGA lung adenocarcinomas samples have a somatic NF1 mutation. NF1-mutated tumors have higher TMB (p < 0.0001), higher expression of immune genes (“hot phenotype”) and higher CD8 + T cell (p = 0.03) and macrophage (p = 0.02) infiltrations compared to NF1 wild-type tumors. NF1 mutation status appears as a candidate predictive biomarker for ICI response in lung adenocarcinoma patients.

We compared the mRNA levels of nine immune genes in NF1-mutated (N = 66; 9.6%) vs NF1 WT (N = 620; 90.4%) lung adenocarcinomas (Fig. 2).We observed a significantly higher expression of CXCL9 (p = 0.008), PD-L1 (p = 0.010), PD-L2 (p = 0.011), and CD8A (p = 0.006) in NF1-mutated samples vs NF1 WT samples.Lung adenocarcinoma samples with a TMB ≥ 10 mut/Mb also had a higher expression of these same genes compared to samples with a TMB < 10 mut/Mb.TP53-mutated samples had a significantly higher expression of all investigated immune markers than TP53 WT samples.Conversely, KRAS-mutated samples showed no increase in the expression of immune genes compared to KRAS WT tumors.KEAP1 and STK11-mutated tumors showed a significant decrease in the expression of the nine immune genes vs WT tumors (Supplementary Table 2).
In a 2023 single-center retrospective study, Wang et al. showed an enhanced proliferation and immune activity of macrophages and NK cells in case of germline NF1 mutations 9 in patients with juvenile myelomonocytic leukemia.Data are available for NF1-mutated sporadic cancerswhich mainly include melanoma.Johnson et al. conducted a study on 65 patients with advanced melanoma treated with ICI.The subgroup of patients with NF1-mutated melanomas had a higher TMB and better response rates (74%) than those with BRAF/NRAS-mutated and wild-type melanomas 5 .Furthermore, a retrospective multicenter analysis revealed a significantly better median OS (p = 0.0154) when receiving first-line immune checkpoint inhibitor treatment for NF1-mutated (n = 80) than for wild-type (n = 432) melanomas 6 .However, there are currently no clinical trials evaluating ICI efficacy that enrolled patients according to a stratified randomization on their NF1 status 2 .In the context of lung adenocarcinomas, only two case reports showed durable responses to ICI in two patients with sporadic NF1-mutated cancers.The first one showed a progression free survival of 95.4 weeks with pembrolizumab 7 .The second reported a stable disease for 8 months in a stage IV lung adenocarcinoma patient treated with pembrolizumab 8 .These observations support the need to explore NF1 as a predictive biomarker of ICI sensitivity in the context of lung adenocarcinoma.
MSK-IMPACT study showed that response to ICI is associated with a high TMB, whatever tumor histological subtype.In lung cancers, an association was identified between an elevated TMB and durable clinical benefits of ICI [10][11][12] .Three studies presented at recent congresses confirmed a significantly higher TMB (P < 0.001, p < 0.0001 and p < 0.0001) and a higher expression of PD-L1 expression (p < 0.01, p = 0.05 and p < 0.0001) in NF1mutated versus NF1-WT lung cancers [13][14][15] .Our in silico analysis of 686 samples confirmed these results: NF1 mutation was associated with a higher TMB (p < 0.0001) and a higher expression of key immune genes.We showed a significant enrichment of NF1-mutated samples in "hot tumors" (p = 0.02) and an increased CD8 + T-cell infiltration (p = 0.03).
Given that NF1-mutated lung adenocarcinoma is frequently comutated (and especially with TP53), one cannot exclude an impact of these co-mutations on the TMB and immune infiltration.However, our multivariate analysis shows that NF1 status is significantly associated (p = 0.0183) with high TMB independently of the co-mutation status.TP53 mutations have been reported to be a biomarker associated with ICI benefit response, in particular in case of KRAS co-mutations 10 .A 2017 clinical study aimed to identify novel biomarkers for immune check-point inhibitor responses in 904 patients with lung adenocarcinoma 16 .Patients with TP53-mutated lung adenocarcinoma were characterized by a significant increased expression of PD-L1 (p < 0.05) and TMB (p < 0.001), and a higher tumor infiltration by CD8 + T cells (p < 0.05).In 2020, a study of 637 patients with non-small cell lung cancer (NSCLC) confirmed a high TMB (p = 0.007) and high PD-L1 expression (p < 0.001) in TP53-mutated samples 10 .NF1 mutation status was not investigated in these studies.Here, we confirmed that TP53 mutations were correlated with higher TMB (p < 0.0001) and higher expression of immune markers.Pilar et al. showed that the combination of multiple biomarkers (PD-L1 immunohistochemistry, T-cell infiltration, and TMB) increased performance, compared with the three biomarkers alone 17 .Our results suggest that a mixed score including NF1 should be considered.
The presence of NF1 mutations was correlated with high TMB.This sole observation did not allow to determine if NF1 mutations could be a consequence of genetic instability.The transcriptional and immune analyses were limited to small datasets.Because of this limited size, we could not check whether the subgroup of NF1-mutated patients with low TMB expressed markers of immune infiltration.In addition, one of the limitations of in silico analysis is the lack of available clinical data.We did not have sufficient clinical outcomes of patients with NF1-mutated lung adenocarcinoma and treated Fig. 1 | TMB in lung adenocarcinoma according to mutation status.a TMB in NF1 mutated lung adenocarcinomas (N = 341 tumors) versus NF1 WT lung adenocarcinomas (N = 3840 tumors) showed a statistically significant difference with a mean TMB at 14.1 mut/Mb in NF1-mutated tumors vs 6.5 mut/ Mb in NF1 WT tumors (p < 0.0001).b NF1-mutated lung adenocarcinomas showed a higher mean TMB vs TP53-, KRAS-, KEAP1-, and STK11-mutated lung adenocarcinomas.Numbers of TP53, KRAS, KEAP1 and STK11 co-mutations for the 341 NF1-mutated samples are available in Supplementary Table 1.***P ≤ 0.001 and ****P ≤ 0.0001.https://doi.org/10.1038/s41698-024-00524-xwith ICI in these TCGA public databases.We were unable to make a multivariate model to predict the efficacy of ICI.We therefore looked for factors associated with a high TMB, that is correlated with durable clinical benefits of ICI.To study the impact of genes on ICI response, a progression free survival analysis would have been necessary.Additional studies are warranted to specify the biological effect of NF1 mutations on immune environment in lung adenocarcinoma.
We showed that NF1-mutated lung adenocarcinomas are associated with a high TMB, high immune genes expression, and CD8 + T-cells infiltration.To date in clinical practice, besides PD-L1 expression that is actually use to drive the choice of immunotherapy regimens in non-small cell lung cancer that lacks a driver mutation, there is no strong predictive factor of immune checkpoint blockade.We suggest the potential of NF1 alterations as a novel biomarker for ICI, warranting further investigations alone or in combination with other ICI predictive biomarkers.Our results highlight the need for clinical trials evaluation ICI efficacy specifically dedicated to NF1mutated lung adenocarcinomas with a randomization based on NF1 status.

Data acquisition
We studied in silico data using 14 publicly available datasets with lung adenocarcinoma samples on the cBioPortal website (The Cancer Genome

Tumor mutational burden and genetic alterations
We analyzed the presence and type of NF1 alterations, the level of NF1 mRNA expression (log2 RNA seq V2 RSEM), and the tumor mutational burden (TMB) defined as the number of mutations per megabase (mut/Mb).

Statistical analyses
Comparisons were performed with Student t-test or Wilcoxon for quantitative variables.ANOVA with false discovery rate correction (two-stage step-up method) was performed for multi-group comparison.Clustering analyses and heatmap visualization were generated using the "Complex-Heatmap" R package.Samples clustering was performed according to the mRNA expression level of significant immune genes.Enrichment score was calculated using the Fisher exact test.We set up univariate and multivariate logistic regression models to determine the factors associated with a high TMB (defined as >10 Mut/Mb).We included as clinical and genomic criteria: age, gender, smoking status (cut-off: 20 pack-years) and the mutational status of NF1, KRAS, TP53, KEAP1 and STK11.All p-values were twosided, and the level of significance was set at p < 0.05.Statistical analyses were performed with R statistical software (version 4.2.2) and GraphPad Prism (version 10.0.0).

Table 1 |
Univariate and multivariate logistic regression analysis of factors associated with a high TMB TMB Tumor Mutational Burden (mut/Mb), OR Odds ratio.